Osteogenic paste compositions and uses thereof

ABSTRACT

Described are osteogenic paste compositions with enhanced osteoinductive properties for use in bone repair. Compositions comprising a quickly resorbable paste carrier, a more slowly resorbed mineral matrix, and Bone Morphogenetic Protein (BMP) or other osteogenic factor are described which enable increased osteoinductive activity while retaining a reliable scaffold for the formation of new bone at the implant site. Methods for making and methods for therapeutic use of the compositions are also disclosed.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/923,117 filed Aug. 6, 2001 which claims the benefit of U.S. PatentApplication Ser. No. 60/118,614 filed Feb. 4, 1999, both of which arehereby incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates generally to osteogenic paste compositionscontaining a paste-form carrier and an osteogenic factor. In onespecific aspect, this invention relates to osteogenic paste compositionscontaining a paste-form carrier, an osteogenic factor, and a substantialmineral component to provide a lasting scaffold for bone growth. Thisinvention also relates to methods of making and using the osteogenicpaste compositions.

BACKGROUND

As further background, bone grafting is commonly used to augment healingin the treatment of a broad range of musculoskeletal disorders. Graftinghas been effective in reconstruction or replacement of bone defects, toaugment fracture repair, to strengthen arthrodeses and to fill defectsafter treatment of tumors. Autograft techniques have been known for over100 years and include the use of cortical and cancellous bone asgrafting material. The use of autografts presents several seriousdrawbacks including the limited amount of potential donor materialavailable, the requirement for two surgical intrusion sites on thepatient, a high incidence of donor site morbidity, the tedious andcomplex nature of the techniques, particularly when vascularized graftsare involved, and the fact that donated bone can rarely be preciselysized or shaped to fit the needs of the implant site. Allografts canalso be used in analogous procedures. Allografts have the benefits ofavoiding two-site surgery on the patient and the elimination of donorsite morbidity risk. However, allografts have increased risks of diseasetransmission and immunogenic implant rejection. Procedures used toreduce these new risks inherently decrease the viability of theallografts as effective implant material. Procedures with allograftsalso remain tedious and complex, suffer from limited source material andhave the same limitations on sizing and shaping the implant to optimallyfit the needs of the implant site.

A number of compositions have been developed to augment or replaceautographic and allographic techniques to reduce or avoid the abovementioned drawbacks. Ceramics such as hydroxyapatite, tricalciumphosphate (TCP), and coralline hydroxyapatite have been shown to bebeneficial osteoconductive matrices for use as fillers and/or expandersin bone graft material. Ceramics can add compression strength, but lackosteoinductive properties and, when used alone, lack shear and tensilestrength. R. W. Bucholz, A. Carlson, R. E. Holmes, Hydroxyapatite andtricalcium phosphate bone graft substitutes. Orthop. Clin. North Am.,Vol. 18(2), 1987, pg. 323-334 and R. W. Bucholz, A. Carlson, R. E.Holmes, Interporous hydroxyapatite as a bone graft substitute in tibialplateau fractures, Clin. Orthop., Vol. 240, 1989, pg. 53-62. Further, ithas been shown in animal studies, that such ceramics can be filled withmarrow to provide a beneficial level of initial progenitor cells andother osteogenic factors. H. Ohgushi, V. M. Goldberg, A. I. Caplan,Heterotopic osteogenesis in porous ceramics induced by marrow cells, J.Orthop. Res., Vol. 7, 1989, pg. 568-578.

The calcium phosphate based ceramics differ widely in their resorptioncharacteristics once implanted. In addition to other factors, theresorption rate tends to increase with surface area of the ceramic,which in turn depends on the ceramic's particle shape, size, density andporosity. TCP is degraded 10-20 times faster than hydroxyapatite. Alsopartly as a result, if new bone development is established with a TCPimplant, the TCP is generally remodeled better than hydroxyapatite inthe final stage of bone formation. It is noteworthy that TCP is resorbedby osteoclast cells, whereas, the much slower resorption ofhydroxyapatite is effected mainly by foreign-body giant cells. The giantcells have a limit as to the amount of hydroxyapatite they will resorb.

Pure ceramics do not offer optimum handling characteristics duringimplantation, but do offer excellent biocompatibility properties andtend to bond well to the existing bone. Ohgushi, et al. teaches the useof marrow infiltration of ceramics, while others have used variousbinders with granulated ceramics to formulate workable pastes thatsolidify to provide stable implants of desired shape and size. C. P.Desilets, L. J. Marden, A. L. Patterson and J. O. Hollinger, Developmentof synthetic bone-repair materials for craniofacial reconstruction, J.Craniofacial Surgery, Vol. 1(3), 1990, pg. 150-153.

Demineralized bone matrix (DBM) preparations have been researchedextensively for use as bone implant material. DBM is prepared throughthe acid extraction of minerals from bone. It includes the collagenmatrix of the bone together with acid insoluble proteins including bonemorphogenic proteins (BMPs) and other growth factors. DBM can beprocessed as crushed granules, powder or chips. It can be formulated foruse as granules, gels, sponge material or putty and can be freeze-driedfor storage. Sterilization procedures required to protect from diseasetransmission may reduce the activity of beneficial growth factors in theDBM. DBM provides an initial osteoconductive matrix and exhibits adegree of osteoinductive potential, inducing the infiltration anddifferentiation of osteoprogenitor cells from the surrounding tissues.DBM lacks structural strength and is therefore only useful to fill wellsupported, stable skeletal defects such as cysts, simple fractures, andfillers for autographs and allographs. Examples of commerciallyavailable DBM products are Grafton® Allogenic Bone Matrix by Osteotech,Shrewbury, N.J., and Dynagraft® by Gensci Regeneration Laboratories,Irvine, Calif.

Various combinations of the above-mentioned bone implant materials havebeen made with a desire to obtain the benefits of the individualcomponents without their individual drawbacks. Some combinations havemet with a measure of success, but Y. Yamazaki, S. Shioda and S. Oida,Experimental Study on the Osteo-Induction Ability of Calcium PhosphateBiomaterials with added Bone Morphogenic Protein, Transaction of theSociety for Biomaterials, 1986, pg. 111, teach that not all combinationsof elements known to be individually beneficial for bone implantmaterials are additive in their beneficial characteristics or effectiveas composite implant materials. Yamazaki, et al. found that theosteoinductive potential of DBM and osteogenic protein extractstherefrom are inhibited by the addition of TCP or hydroxyapatite. Noosteogenic composition has yet been found to be optimum in generalizedusage and clinical results vary widely, even with seemingly well definedcompositions. There remains a need for improved osteogenic implantmaterials that are consistently strongly osteoinductive,osteoconductive, easily workable in surgical procedures, and thatprovide strength and stability for new bone formation during the earlystages of bone development, but are essentially completely incorporatedand remodeled into bone by the end of the osteogenic process.

Compositions of mixed ceramics of TCP/hydroxyapatite and collagen arecommercially available and can be enhanced by filling with autogenousbone marrow prior to implant. The composites are available as pastes orsoft strips and tend to flow away from the implant site. The implantmust therefore be carefully retained in place until the composite andany surrounding bleeding has fully clotted.

Compositions of bone gel known as GRAFTON® (see U.S. Pat. No. 5,481,601)comprising glycerol and DBM have been used singly and mixed withsand-like powder. Such compositions have been used to fill bone voids,cracks and cavities. GRAFTON® is available in flexible sheets or as aputty, thus making the composition more easily workable duringimplantation. Again, such compositions tend to flow away from theimplant site.

Jefferies, in U.S. Pat. Nos. 4,394,370 and 4,472,840, teach a boneimplant material composition of collagen and DBM or solubilized BMP thatis optionally crosslinked with glutaraldehyde.

Caplan et al., in U.S. Pat. No. 4,620,327, describe the combination andpartial immobilization by chemical cross-linking of soluble boneproteins with a number of solids to be implanted for bonerepair/incorporation, including xenogenic bony implants, allografts,biodegradable masses and prosthetic devices to enhance new bone orcartilage formation. Ries et al., in U.S. Pat. No. 4,623,553, describethe glutaraldehyde or formaldehyde cross-linking of collagen andhydroxyapatite or TCP. Ries does not include any osteoinductive elementsand is deemed only osteoconductive.

Some researchers have suggested the use of composites of TCP and/orbiopolymers like polylactide, polyglycolide or their copolymers andparticulate bone derivatives or BMP for craniofacial reconstruction. TheTCP and biopolymers would provide a scaffold for new bone formation. Thebone derivatives and BMP would induce osteogenesis beyond the slow,shallow osteoconduction induced by TCP and biopolymers alone. Desilets,et al.

Jefferies, in PCT WO 89/04646, describes osteoinductive implantmaterials having increased tensile strength by surface activating DBM orBMP with gluteraldehyde or other suitable cross-linking agent, followedby addition to a porous solid matrix where the activated DBM or BMPreacts with the solid to increase the cohesive strength of thecomposite. Jefferies also teaches the incorporation of therapeutic drugsinto the matrix for the slow beneficial release thereof during thecourse of treatment.

In light of this background, there remain needs for improved osteogeniccompositions and methods that effectively induce and support bone growthin mammals, including humans. The present invention is addressed tothese needs.

SUMMARY OF THE INVENTION

The present invention generally provides osteogenic paste compositionsincluding a paste-form carrier such as a gelatin paste and at least oneosteogenic factor such as BMP-2 or another similar bone morphogeneticprotein. A particular feature of the present invention relates to thediscovery that the inclusion of an osteoblast- andosteoclast-stimulating osteogenic factor in a paste-form compositionincluding a resorbable paste carrier causes a rapid and prematureresorption of the carrier. This rapid resorption of the carrier candiminish or eliminate the capacity of the paste-form composition toeffectively stimulate and support new bone formation in a void filledwith the composition. This is particularly the case in primates,including humans, in which the rate of new bone formation is relativelyslow.

Accordingly, one preferred embodiment of the present invention providesan osteogenic paste composition effective for the induction and supportof new bone growth in a primate. The implant composition comprises aresorbable paste-form carrier, including for instance a paste made witha substance such as gelatin, hyaluronic acid, and/or carboxymethylcellulose. The composition also includes an effective amount of anosteogenic factor, such as a bone morphogenetic protein, that stimulatesboth osteoblast cells and osteoclast cells. In addition, compositionincludes a substantial proportion of a particulate mineral that iseffective to provide a scaffold for bone ingrowth when the resorbablepaste carrier is resorbed at an increased rate due to the stimulation ofosteoclast cell activity. Preferred such compositions of the inventionare provided wherein the resorbable paste carrier includes gelatin,and/or wherein the resorbable paste carrier is flowable at temperaturesabove the body temperature of the mammal in which it is to be implanted,but transitions to a relatively non-flowable mass at or slightly abovesaid body temperature.

Importantly, the particulate mineral matrix constitutes a substantialproportion of the paste composition as a whole, in order to provide aneffective scaffold for bone ingrowth. In most cases, the particulatemineral have an average particle size between about 0.050 and about 5.0mm, and will constitute about 20% to 80% by volume of the overallcomposition, more typically higher levels of about 40% to about 80% byvolume. The particulate mineral can include, for example, a natural orsynthetic mineral, e.g. a material selected from the group consisting ofbone particles, Bioglass®, tricalcium phosphate, hydroxyapatite,biphasic calcium phosphate, corraline hydroxyapatite, biocompatibleceramic and non-resorbable biocompatible organic polymer. Biphasiccalcium phosphate is a particularly preferred synthetic ceramic for usein the present invention, advantageously having a tricalciumphosphate:hydroyapatite weight ratio of about 80:20 to about 90:10.

In some preferred modes of carrying out the invention, the mineralcomprises cancellous or cortical bone particles having an averageparticle size between about 0.050 and about 5.0 mm. Such bone particlescan be of human or non-human (e.g. bovine) origin. In other modes, themineral comprises tricalcium phosphate, biphasic calcium phosphate orhydroxyapatite having a particle size of about 0.50 to about 5.0 mm. Instill another aspect of the invention, the paste composition furthercomprises demineralized bone matrix. The weight ratio of demineralizedbone matrix to resorbable carrier is preferably between about 1:4 andabout 3:2, respectively.

In one particularly preferred form of the present invention, anosteogenic paste composition for the induction of new bone growth in aprimate is provided, comprising:

(a) a resorbable paste carrier comprising gelatin, the resorbable pastecarrier formulated to be flowable at temperatures above the bodytemperature of the primate, and to transitions to a non-flowable mass atsuch body temperature;

(b) demineralized bone matrix;

(c) a bone morphogenic protein that stimulates osteoblasts andosteoclasts, more preferably BMP-2 or BMP-7; and

(d) cortical or cancellous bone particles, having an average particlesize of between about 0.050 and about 5.0 mm, and constituting about 20%to about 80% by volume of the overall implant composition.

Still other preferred embodiments of the present invention providemethods for treating bone trauma, defect or disease, or for effectingartificial arthrodeses in a mammal, comprising the step of implanting anosteogenic paste composition of the invention in a primate at a site ofdesired new bone formation.

The present invention provides an improved osteogenic implant materialthat is strongly osteoinductive and that can be formed into preciseshapes either prior to implant or during the surgical procedure itself.The present invention also provides implant materials that retain stableshapes at the implant site and do not deform, migrate, or flow away fromthe implant site before ossification is established. Significantly, thepresent invention also provides advantageous implant materials that haveenhanced osteoinductive potential and provide a matrix that is workableduring implantation, but not resorbed prior to the establishment of bonewithin the void to be filled. Such preferred compositions provide amineral scaffold for the generation of new bone that is subsequentlyincorporated into the bone matrix as the new bone matures. The inventionalso provides methods for preparing such compositions and of using suchcompositions to treat bone trauma, disease and defects, whereverosteogenesis is desired. These and other objects, features andadvantages of the present invention will be readily apparent from thefollowing description.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows ex vivo alkaline phosphatase activity as a function of timefor intramuscular rat implants of demineralized bone matrix, a paste ofgelatin and demineralized bone matrix, and of rhBMP-2 in each of acollagen sponge, a paste of gelatin and demineralized bone matrix, andin a paste of gelatin alone.

FIG. 2 shows calcium content of explanted ossicles as a function of timefor intramuscular rat implants of demineralized bone matrix, a paste ofgelatin and demineralized bone matrix, and of rhBMP-2 in each of acollagen sponge, a paste of gelatin and demineralized bone matrix, andin a paste of gelatin alone.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to certain embodiments thereof andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations, further modificationsand applications of the principles of the invention as illustratedherein being contemplated as would normally occur to one skilled in theart to which the invention relates.

As indicated above, the present invention generally provides osteogenicpaste compositions including a paste-form carrier and abone-growth-inducing amount of an osteogenic factor such as a bonemorphogenetic protein (BMP). The present invention features osteogenicpaste compositions effective for use in primates, wherein thecompositions exhibit high osteoinductive potential and provide a lastingmineral scaffold to support bone ingrowth. Such preferred compositionsinclude a resorbable paste-form carrier, for example an aqueous pasteincluding gelatin, and an osteogenic factor that stimulates the actionof both osteoblasts (which biologically facilitate the formation ofbone) and osteoclasts (which biologically facilitate the resorption ofbone). In accordance with the present invention, it has been found thatthe incorporation of effective inductive amounts of such osteogenicfactors, for example bone morphogenetic proteins, stimulates osteoclaststo such a level that the resorbable carrier is too quickly resorbed and,in the absence of a high mineral component in the composition, causesthe performance of the composition to suffer in some cases to the extentthat the observance of substantial bone ingrowth is sporadic.

Accordingly, a feature of the present invention is the provision of apaste-form osteogenic composition that includes a substantial amount ofa relatively slowly-resorbed mineral component that remains at theimplant site after the carrier has been rapidly resorbed, in order toprovide a scaffold for new bone formation that is not prematurelyresorbed due to the osteoclastic potentiation by the bone morphogenicprotein in the composition. The present invention also provides methodsfor using such osteogenic compositions in treatment of bone trauma,disease and defects, for artificial arthrodeses and for other treatmentwhere new bone formation is desired, especially in primates, includinghumans.

Generally speaking, compositions in accordance with the presentinvention are in paste form and comprise a resorbable carrier,especially a gelatin paste, and an osteogenic factor such as a BMP thatstimulates osteoblasts and osteoclasts, e.g. BMP-2 or BMP-7, especiallyBMP-2. The preferred compositions of the invention also include asubstantial proportion (i.e. at least about 20% by volume) of aparticulate, porous mineral matrix dispersed within the carrier. Suchcompositions can also include other resorbable components, for exampledemineralized bone matrix.

As to the carrier, in accordance with the present invention, it will bebiologically resorbable and will contribute to providing a paste form tothe composition allowing its implantation and retention at the candidatesite for bone ingrowth. Preferred carriers will include resorbablemacromolecules from biological or synthetic sources, for examplegelatin, hyaluronic acid carboxymethyl cellulose, collagen, peptides,and the like. In more preferred inventive forms, the resorbable carrier,especially gelatin, is formulated into the composition such that thecomposition is flowable at temperatures above the body temperature ofthe mammal into which the material is to be implanted, but transitionsto be relatively non-flowable at or slightly above such bodytemperature. The resorbable carrier may be formulated into thecomposition so the flowable state is a liquid or a flowable gel, and thenon-flowable state is a stable gel or solid. In certain embodiments ofthe invention, the resorbable carrier is gelatin derived from thespecies receiving the implant, while in others the gelatin is derivedfrom a species other than that of the mammal receiving the implant.

As is well known, when gelatin is solubilized in warm or hot aqueoussolutions, the molecules have little organization. However, as a gelatinsolution is allowed to cool, the gelatin molecules intertwine into athree dimensional matrix and the viscosity of the solution increases. Ata characteristic set temperature, a phase transformation changes theflowable solution into a non-flowable gel. The set time, set temperatureand density of the resulting non-flowable mass are dependent on severalfactors including the concentration of gelatin, the molecular weight andthe intrinsic viscosity of the gelatin molecules and on the pH of thecomposition. Other components of the composition can affect the set timeand temperature as well. The shortest set times are typically at theisoelectric point of the gelatin molecules.

As indicated in the passages above, other carriers can be used insteadof or in addition to gelatin, to provide the paste composition.Illustrative macromolecules for these purposes include, for example,hyaluronic acid, cellulose derivatives such as carboxymethyl cellulose,collagens, peptides and the like. These and other similar materialswhich function as resorbable thickening agents will be suitable, andtheir incorporation into compositions of the present invention will bewithin the purview of those ordinarily skilled in the field given theteachings herein.

Paste compositions of the invention may also include other potentiallyosteoinductive substances, including for example demineralized bonematrix (DBM). As is known in the field, DBM can be prepared by aciddemineralization of bone and when so prepared contains, among otherconstituents, the collagen matrix of the bone and acid insolubleproteins. DBM has been shown previously to be mildly osteoinductive byitself and has a favorable porous matrix for the ingrowth of bone.Methods of producing DBM are known in the art and are, therefore notelaborated upon here (see for example U.S. Pat. No. 5,405,390, hereinincorporated by reference for this purpose). In a preferred form, DBMhaving a particle size of between about 0.10 and about 1.00 mm will beincorporated within compositions of the present invention. The DBM canbe derived from the same or a different mammalian species as that inwhich the implant material is to be used. When used, the DBM ispreferably blended with the resorbable carrier in a weight ratio betweenabout 1:4 and about 3:2 DBM to resorbable carrier. Commerciallyavailable preparations of DBM are suitable for use in the presentinvention provided they may be uniformly blended with the other elementsof the composition.

As indicated above, preferred paste compositions of the invention alsoinclude an osteoinductive factor, such as an osteoinductive protein or anucleotide sequence encoding an osteoinductive protein operablyassociated with a promoter (e.g. provided in a vector such as a viralvector), for example a bone morphogenetic protein or a gene encoding thesame operationally associated with a promoter which drives expression ofthe gene in the animal recipient to produce an effective amount of theprotein. The bone morphogenic protein (BMP) in accordance with thisinvention is any BMP able to stimulate differentiation and function ofosteoblasts and osteoclasts. Examples of such BMPs are BMP-2, BMP-4, andBMP-7, more preferably rhBMP-2 or rhBMP-7, most preferably, rhBMP-2.Purified recombinant BMPs are preferred for use in the inventivecompositions for their provision of high osteoinductive potentials. BMPgene sequences and methods for producing recombinant andnaturally-derived BMPs are known in the art, and for additionalinformation on this subject reference may be made, for instance, to U.S.Pat. Nos. 5,108,753; 5,187,076; 5,366,875; 4,877,864; 5,108,922;5,116,738; 5,013,649; 5,106,748; and 4,294,753; and InternationalPublication Nos. WO93/00432; WO94/26893; and WO94/26892. Theosteoinductive factor may also be LIM mineralization protein (LMP) or asuitable vector incorporating a gene encoding the same operablyassociated with a promotor, as described in WO99/06563 (see also genbankaccession No. AF095585). When such vectors are employed as osteogenicfactors in accordance with the invention, they are preferably deliveredin conjunction with cells, for example autologous cells from therecipient of the implant. Most preferably the vector is delivered inconjunction with autologous white blood cells derived from bone marrowor peripheral blood of the recipient.

The osteogenic factor will be incorporated in an amount which iseffective to stimulate the formation of bone within the animalrecipient. In more preferred compositions incorporating proteinosteogenic factors, the osteogenic factor will be incorporated in aweight ratio of about 1:100 to about 1:1000 relative to the overallcomposition, more preferably about 1:100 to about 1:500. As will beunderstood, when the osteogenic factor comprises a nucleotide sequence,sufficient amounts of the delivery vehicle (vector) will be incorporatedto cause significant transduction of cells, so as to cause thegeneration of sufficient protein at the site to induce bone formation.

The osteogenic factor may be incorporated into the paste in any suitablemanner, for example by pre-impregnating the mineral particles with theosteogenic factor prior to blending with the paste carrier, by blendingthe factor with the carrier, or both. Alternatively or in addition,amounts of the osteogenic factor can be blended with the carrier/mineralmixture immediately prior to implantation.

The porous mineral used in accordance with the preferred embodiments ofthe present invention includes a natural or synthetic mineral that iseffective to provide a scaffold for bone ingrowth as the resorbablecarrier and other more rapidly resorbed elements of the implantcomposition are resorbed. Illustratively, the mineral matrix may beselected from one or more materials from the group consisting of boneparticles, Bioglass®, tricalcium phosphate, biphasic calcium phosphate,hydroxyapatite, corraline hydroxyapatite, and biocompatible ceramics.Biphasic calcium phosphate is a particularly preferred synthetic ceramicfor use in the invention. Desirably, such biphasic calcium phosphatewith have a tricalcium phosphate:hydroxyapatite weight ratio of about50:50 to about 95:5, more preferably about 70:30 to about 95:5, evenmore preferably about 80:20 to about 90:10, and most preferably about85:15.

In another preferred aspect of the invention, the mineral matrixincludes bone particles, possibly cancellous but preferably cortical,ground to provide an average particle diameter between about 0.050 and5.0 mm. Both human and non-human sources of bone are suitable for use inthe instant invention, and the bone may be autographic, allographic orxenographic in nature relative to the mammal to receive the implant.Appropriate pre-treatments known in the art may be used to minimize therisks of disease transmission and/or immunogenic reaction when usingbone particles in the mineral matrix.

In one embodiment of the instant invention, xenogenic bone that has beenpretreated to reduce or remove its immunogenicity is used to provide theporous mineral matrix in the implant composition. For example, calciningor deproteinizing of the bone can be used to reduce the risks ofimmunogenic reactions to the implant material.

The level at which the mineral is incorporated into the preferredmineral-enhanced compositions of the invention is important to theprovision of beneficial osteoinductive properties to the compositions.In general, the minimum level of mineral is dependent on activity of theBMP in the composition; the higher the activity of the BMP, the greaterthe content of the mineral matrix required to counter the osteoclasticpotentiation of the BMP. As the BMP concentration increases, so does therate of resorption of the resorbable carrier and DBM if present. As aresult, the mineral content must be sufficient to provide a scaffold forthe ingrowth of new bone while not abrogating the structural integrityof the composition. The mineral should also be such that as the new bonematures, the mineral is made an integral part of the tissue matrix or isresorbed during remodeling of the new bone in the natural course of bonegrowth and development.

In a preferred form of the invention, the mineral constitutes about 20%to about 80% by volume of the composition, more preferably about 40% toabout 80%. Generally speaking, the amount of mineral in the pastecomposition will be sufficient to provide a scaffold that will remain inthe patient for a period of time sufficient for the formation of osteoidin the area for which bone growth is desired. Typically, this period oftime will be about 6 to about 8 weeks.

As further enhancements of the compositions of the present invention,those skilled in the art will readily appreciate that other osteogenicenhancing factors may be incorporated into the composition. Suchadditional factors include, but are not limited to host compatibleosteogenic progenitor cells, autographic bone marrow, allographic bonemarrow, transforming growth factor-beta, fibroblast growth factor,platlet derived growth factor, insulin-like growth factor,microglobulin-beta, antibiotics, antifungal agents, wetting agents,glycerol, steroids and non-steroidal anti-inflammatory compounds.

In use, the paste-form implant compositions of the invention areimplanted at a site at which bone growth is desired, e.g. to treat adisease, defect or location of trauma, and/or to promote artificialarthrodesis. The paste form of the compositions enables theirpositioning, shaping and/or molding within voids, defects or other areasin which new bone growth is desired. In the case of implant compositionswhich are flowable at temperatures higher than the body temperature ofthe mammal in which they are to be implanted, yet which transition to anon-flowable mass at or near such body temperature, the composition isheated to a temperature at which it is flowable, but which will notdenature any osteogenic factor present; molded or otherwise shaped tothe shape of the desired new bone; cooled to a temperature sufficient totransition the osteogenic implant material into a non-flowable masseither in situ or implanted at the site of desired new bone formationafter setting up. In other preferred situations, the paste compositiondoes not require heating to above body temperature (about 37° C.) forflowability, for example wherein the paste composition is flowable attemperatures below 37° C. and cures or solidifies into a non-flowablemass upon heating or upon contact with a separate curing agent. Suchcases are particularly advantageous in that heat-induced denaturation ofthe osteogenic factor is less of a concern.

Once in place, the paste form implant compositions of the invention willeffectively induce and support the ingrowth of bone into the desiredarea even in a primate subject such as a human exhibiting a relativelyslow rate of bone formation compared to smaller mammals, for examplerodents or rabbits. In particular, while the paste carrier is generallyresorbed relatively quickly, the substantial mineral component remainsas a scaffolding to support new bone growth in and through the desiredarea. In this regard, it is preferred that the mineral matrix be chosenand included in an amount which will provide a scaffold which isdetectable in the treated subject for a period sufficient for theformation of osteoid in the volume to be filled with bone, typicallyabout 6 to about 8 weeks. This will facilitate effective bone formationeven where the resorbable carrier and other quickly-resorbed componentsof the paste are rapidly eliminated from the implant site.

Compositions of the invention are especially advantageous when used inbones or bone portions that are vascularized to only moderate or lowlevels. These areas present particularly low rates of bone formation,and as such the rapid resorption of the carrier poses enhanceddifficulties. Examples of moderate or only slightly vascularized sitesinclude, for example, transverse processes or other posterior elementsof the spine, the diaphysis of long bones, in particular the middiaphysis of the tibia, and cranial defects

An especially preferred use of paste compositions of the invention is asan implant to promote arthrodesis between vertebrae in spinal fusions inhumans or other primates, including for example interbody, posteriorand/or posterolateral fusion techniques. Although the rate of boneformation in the primate spine is relatively slow overall and thus willbenefit generally from the present invention, the elements to be fusedin posterior and posterolateral fusions exhibit particularly low levelsof vascularization and thus fusions of these elements are expected tobenefit markedly from the invention. In addition, in accordance withother aspects of the invention, the osteogenic paste compositions of theinvention can be incorporated in, on or around a load-bearing (e.g.having a compressive strength of at least about 10000 N) implant devicesuch as a fusion cage, dowel, or other device having a pocket, chamberor other cavity for containing an osteogenic composition, and used in aspinal fusion such as an interbody fusion.

The invention will now be more particularly described with reference tothe following specific Examples. It will be understood that theseExamples are illustrative and not limiting of the invention.

EXAMPLE 1

Rat Study Comparing the Effect of rhBMP-2 on Osteogenic Capacity of aMatrix Consisting of Collagen Derived Gelatin and Demineralized BoneMatrix (DBM).

Thirty young adult male Sprague-Dawley rats weighing between 200-220 g,were randomly assigned to two groups. Each animal was surgicallyimplanted with six 0.050 mL samples. The samples were inserted inpockets incised into the rectus abdominus muscles on either side of themidline. Samples were placed three to a side, evenly spaced in linesextending from below the sternum to above the mid-groin.

Two of the six samples for each animal were positive controls, one beingDBM alone, the second being Helistat® Absorbable Collagen Sponge (ACS)onto which 0.004 mg rhBMP-2 had been adsorbed. Group I animals were alsogiven duplicate samples of a gelatin/DBM injectable matrix (Gelatin BonePaste) and duplicate samples of the Gelatin Bone Paste mixed with 0.001mg rhBMP-2. Group II animals were given duplicate samples of the GelatinBone Paste mixed with 0.002 mg rhBMP-2 and duplicate samples of thegelatin without DBM (Gelatin) mixed with 0.002 mg rhBMP-2.

Five animals from each group were sacrificed at each time point of two,fourteen and twenty-one days. At sacrifice, the implant areas wereexcised and analyzed for alkaline phosphatase activity, radiography,bone density, histology and histomorphometry.

FIG. 1 shows alkaline phosphatase activity in the samples. Increasedactivity is indicative of infiltration of the implants byosteoprogenitor cells. The timing and magnitude of increased activity isevidence of the osteoinductive potential of the implant. The GelatinBone Paste samples with rhBMP-2 exhibited earlier and higher alkalinephosphatase activity peaks than the controls or the Gelatin Bone Pastealone. Surprisingly, the 0.001 mg rhBMP-2 samples gave higher activitypeaks than did the 0.002 mg rhBMP-2 samples.

FIG. 2 shows the calcium content of the excised implants. Increasedcalcium content is indicative of bone formation. As with the alkalinephosphatase data, the Gelatin Bone Paste samples with rhBMP-2out-performed both the controls and the Gelatin Bone Paste withoutrhBMP-2 samples in initiating calcification of the implant. It is againsurprising that the 0.001 mg rhBMP-2 in Gelatin Bone Paste samples weremore effective at initiating calcification than the 0.002 mg rhBMP-2 inGelatin Bone Paste samples. It is also noteworthy that the twenty-oneday samples of the higher concentration of rhBMP-2 showed a diminishingof calcification compared to the fourteen day samples.

These data are indicative of the osteoclastic potentiation of rhBMP-2balancing its osteoblast stimulating properties; higher rhBMP-2concentrations stimulate the resorption of the essentially collagenmatrix, limiting the osteogenic potential of such matrices that do notincorporate mineral matrix elements to provide prolonged scaffolding forthe bone formation process. It should be noted that the ACS controlscontaining 0.004 mg rhBMP-2 and the gelatin samples containing 0.002 mgrhBMP-2 had the most readily resorbable matrices and gave the poorestcalcification performances for samples containing rhBMP-2. See FIG. 2.

EXAMPLE 2

Monkey Study Comparing Osteogenicity of rhBMP-2 Containing ImplantMatrices.

Studies in a monkey spinal fusion model were conducted to determine theeffectiveness of three paste compositions. The compositions were thegelatin bone paste of Example 1, that paste containing autograft bonechips, and that paste containing rhBMP-2 at a single level of the spine.Each composition was used in bilateral fusion of vertebra in rhesusmonkeys and analyzed for its ability to induce new bone formation. Indoing so, CT scans were taken every two months over a six-month period.The results demonstrated variable bone growth in monkeys receiving thepaste of Example 1 alone and in the paste containing autograft bonechips, but no growth in monkeys receiving the paste and rhBMP-2. Thisobservation is expected to be due to the premature resorption of thecarrier in the rhBMP-2-containing paste, leaving no matrix for boneingrowth. Accordingly, incorporation of a substantial mineral componentin a BMP-containing paste in accordance with the present invention willprovide a lasting matrix and scaffold for bone ingrowth, thus improvingperformance.

The invention has been described above in detail, with specificreference to its preferred embodiments. It will be understood, however,that a variety of modifications and additions can be made to theprocedures disclosed without departing from the spirit and scope of theinvention. Such modifications and additions are desired to be protected.In addition, all publications cited herein are indicative of the levelof skill in the relevant art, and are each hereby incorporated byreference each in their entirety as if individually incorporated byreference and fully set forth.

1. An osteogenic paste composition effective for the induction of newbone growth in a primate, comprising: a resorbable paste carrier; anosteogenic factor; and a porous particulate mineral in an amount of atleast 20% by volume of the composition, said amount being effective toprovide a scaffold for bone ingrowth as the resorbable paste carrier isresorbed.
 2. The composition of claim 1 which further comprisesdemineralized bone matrix.
 3. The composition of claim 2 wherein theratio of demineralized bone matrix to resorbable carrier is betweenabout 1:4 and about 3:2 by weight.
 4. The composition of claim 2 whereinthe composition comprises 5-45% by weight resorbable carrier.
 5. Thecomposition of claim 1 wherein the resorbable carrier is flowable attemperatures above the body temperature of the mammal, but transitionsto a non-flowable mass at or slightly above said body temperature. 6.The composition of claim 1 wherein the mineral is selected from thegroup consisting of bone particles, bioglass, tricalcium phosphate,hydroxyapitite, corraline hydroxyapitite, biocompatible ceramic andnon-resorbable biocompatible organic polymer.
 7. The composition ofclaim 1 wherein the mineral comprises tricalcium phosphate, biphasiccalcium phosphate, or hydroxyapatite particles having an averageparticle diameter of about 0.050 to about 5.0 mm.
 8. The composition ofclaim 1 wherein the mineral comprises mammalian bone particles having aparticle size of about 0.050 to about 5.0 mm.
 9. The composition ofclaim 1 wherein the mineral comprises cortical human bone particleshaving an average particle diameter of about 0.050 to about 5.0 mm. 10.The composition of claim 1 wherein the osteogenic factor comprises abone morphogenic protein selected from BMP-2, BMP-4, BMP-6 or BMP-7, aLIM mineralization protein, or a nucleotide sequence encoding said bonemorphogenic protein or LIM mineralization protein.
 11. The compositionof claim 1 further comprising one or more osteogenic enhancing factorsselected from the group consisting of osteogenic progenitor cells,autographic bone marrow, allographic bone marrow, transforming growthfactor-beta, fibroblast growth factor, platlet derived growth factor,insulin-like growth factor, microglobulin-beta, antibiotics, antifungalagents, wetting agents, glycerol, steroids and non-steroidalanti-inflammatory compounds.
 12. The composition of claim 1 wherein themineral constitutes about 20% to about 80% by volume of the composition.13. An osteogenic implant material effective for the induction of newbone growth in a mammal, comprising: a resorbable paste carriercomprising gelatin, the resorbable carrier formulated to be flowable attemperatures above the body temperature of the mammal, and to transitionto a non-flowable mass at said body temperature; demineralized bonematrix; an osteogenic factor; and a particulate mineral having anaverage particle size of about 0.050 to about 5.0 mm, said mineralconstituting at least 20% by volume of said composition.
 14. Thecomposition of claim 13 wherein the mineral constitutes about 20% toabout 80% by volume of the composition.
 15. The composition of claim 13wherein the mineral comprises human bone particles.
 16. The compositionof claim 13 wherein the mineral comprises non-human bone particles, saidparticles having been treated to reduce their immunogenicity in humans.17. The composition of claim 13 wherein the osteogenic factor is a bonemorphogenic protein selected from BMP-2, BMP-4, BMP-6 and BMP-7, a LIMmineralization protein, or a nucleotide sequence encoding said bonemorphogenic protein or LIM mineralization protein.
 18. A method forinducing bone growth in a primate, comprising implanting in the primatea composition according to claim 1, at a site at which bone growth isdesired.
 19. The method of claim 18, wherein the site is in the spine ofthe primate.
 20. The method of claim 19, which is a spinal fusion. 21.The method of claim 20, wherein the spinal fusion is an interbody spinalfusion.
 22. The method of claim 20, which is a posterolateral spinalfusion.
 23. The method of claim 19, wherein the primate is a human. 24.The method of claim 20, wherein the fusion includes a fusion betweentransverse processes of adjacent vertebrae.
 25. A method of performing aspinal fusion in a human, comprising implanting between adjacentvertebrae to be fused an effective amount of a composition according toclaim
 1. 26. The method of claim 25, wherein the composition isimplanted in combination with a load bearing device.
 27. A method forinducing bone growth in a primate, comprising: heating an effectiveamount of an osteogenic paste composition to a temperature at which itis flowable, said osteogenic implant material comprising a resorbablepaste carrier that is flowable at temperatures above the bodytemperature of the primate, but which transitions to a non-flowable massat or slightly above said body temperature; an osteogenic factor thatstimulates osteoblasts and osteoclasts; and, a particulate mineraleffective to provide a scaffold for bone ingrowth as the resorbablecarrier is resorbed, said mineral constituting at least 20% by volume ofthe paste composition; implanting said osteogenic paste composition at asite of desired new bone formation; and cooling the osteogenic pastecomposition to a temperature sufficient to transition the osteogenicpaste composition to a non-flowable mass.
 28. The method of claim 27wherein the implant material further comprises demineralized bonematrix.
 29. The method of claim 27 wherein the primate is a human.